Production of human parathyroid hormone from microorganisms

ABSTRACT

The invention provides recombinant plasmids containing in DNA sequences coding for human preproparathyroid hormone. The invention further provides microorganisms, for example  E. coli,  transformed by these plasmids. The invention also provides a plasmid for insertion into yeast and a transformed yeast in which the plasmid contains DNA coding for parathyroid hormone. Parathyroid hormone is then secreted by the transformed yeast. Further the invention provides alternate polypeptides having parathyroid hormone activity, including PTH analogs, fragments and extensions, and provides alternate leader sequences and secretion signal sequences which can be used in the present invention. Finally, there are provided methods for purification of the secreted PTH hormone and/or derivatives.

[0001] This is a Divisional application of prior application Ser. No.08/087,471, filed on Jul. 2, 1993, which is a File Wrapper Continuationof Ser. No. 07/821,478, filed on Jan. 15, 1992, which is a Continuationof Ser. No. 07/404,970, filed on Sep. 8, 1989, now abandoned, which is aContinuation-In-Part of Ser. No. 07/393,851, filed on Aug. 14, 1989,which issued as U.S. Pat. No. 5,010,010 on Apr. 23, 1991, whichapplication, in turn, is a File Wrapper Continuation of Ser. No.06/921,684, filed on Oct. 22, 1986, abandoned.

FIELD OF THE INVENTION

[0002] This invention relates to genetically engineered microorganismscontaining DNA coding for human preproparathyroid hormone.

BACKGROUND OF THE INVENTION

[0003] This application is a continuation-in-part of application Ser.No. 07/393,851 filed Aug. 14, 1989, which is a continuation ofapplication Ser. No. 06/921,684 filed Oct. 22, 1986, now abandoned.

[0004] A number of proteins and peptides that are normally synthesizedby mammalian cells have proven to have medical, agricultural andindustrial utility. These proteins and peptides may be of differentmolecular size and have a number of different functions, for example,they may be enzymes, structural proteins, growth factors and hormones.In essence both proteins and peptides are composed of linear sequencesof amino acids which form secondary and tertiary structures that arenecessary to convey the biological activity. Human parathyroid hormonehas a relatively small molecular. weight, which has made it possible tosynthesize the peptide chemically by the sequential addition of aminoacids. Thus, parathyroid hormone is commercially available, but in verysmall quantities at high cost. As a result, there is no humanparathyroid hormone available at a reasonable price to supply the manypotential medical, agricultural and industrial applications.

[0005] During the past ten years, microbiological techniques employingrecombinant DNA have made it possible to use microorganisms for theproduction of species-different peptides. The microorganism is capableof rapid and abundant growth and can be made to synthesize the foreignproduct in the same manner as bacterial peptides. The utility andpotential of this molecular biological approach has already been provenby microbiological production of a number of human proteins that are nowavailable for medical and other uses.

[0006] Parathyroid hormone (PTH) is one of the most important regulatorsof calcium metabolism in mammals and is also related to several diseasesin humans, animals, e.g. milk fever, acute hypocalsemia and otherwisepathologically altered blood calcium levels. This hormone therefore willbe important as a part of diagnostic kits and will also have potentialas a therapeutic in human and veterinary medicine.

[0007] The first synthesis of DNA for human preproparathyroid hormonewas described by Hendy, G. N., Kronenberg, H. M., Potts, Jr. J. T. andRich, A. 78 Proc. Natl. Acad. Sci. 7365-7369 (1981). DNA complementaryin sequence to PTH mRNA was synthesized and made double stranded (Hendyet al. supra). This cDNA was cloned in pBR 322 DNA and E. coli 1776 wastransfected. Of the colonies with correct antibiotic resistance, 23 outof 200 clones were identified as containing specific human PTH cDNAinserts. However, none of the 23 human PTH clones contained the fulllength insert (Hendy et al., supra). Later Breyel, E., Morelle, G.,Auf'mkolk, B., Frank, R., Blocker, H. and Mayer, H., Third EuropeanCongress on Biotechnology, 10-14 Sep. 1984, Vol. 3, 363-369 describedthe presence of the human PTH gene in a fetal liver genomic DNA libraryconstructed in the phage Charon 4A. A restriction enzyme fragment of thePTH gene was recloned and transfected into E. coli.

[0008] However, the work of Breyel, supra, demonstrated that E. colidegrades human PTH. Thus, a microorganism which shows a stableproduction of intact human parathyroid hormone has so far not beendescribed. Further, parathyroid hormone has never before been isolatedfrom yeast.

SUMMARY OF THE INVENTION

[0009] Accordingly, it is an object of the present invention to providea plasmid containing DNA coding for human preproparathyroid hormone(hPTH) for insertion in Escherichia coli. It is another object of thepresent invention to provide a genetically engineered E. coli containingDNA coding for human preproparathyroid hormone.

[0010] A further object of the present invention is to provide a plasmidfor insertion in yeast containing DNA coding for parathyroid hormone(“PTH”), It is also an object of the present invention to provide atransformed yeast containing DNA coding for parathyroid hormoneincluding human parathyroid hormone, and from which transformed yeast,parathyroid hormone may be obtained.

[0011] Another object of the present invention is to provide newpolymers having parathyroid hormone activity including PTH fragments,extension and analogs. Yet another object is to provide alternate leadersequences and secretion signal sequences which can be used in thepractice of the present invention.

[0012] A still further object of the invention is to provide downstreamprocess technology for purification of intact PTH, as well aspurification of analogs, fragments and extensions.

[0013] Other objects and advantages of the present invention will becomeapparent as the description thereof proceeds.

[0014] In satisfaction of the foregoing objects and advantages, there isprovided by the present invention a novel plasmid for insertion in E.coli, containing DNA coding for human preproparathyroid hormone. Theplasmid when inserted into E. coli functions to transform the E. colisuch that the E. coli then produces multiple copies of the plasmid andthus of the CDNA coding for human preproparathyroid hormone. The plasmidfor human insertion into E. coli of the present invention and thus thetransformed E. coli are distinguishable over prior art plasmids andmicroorganisms, for example as described in Hendy et al., supra, in thatthe plasmid of the present invention contains a double start codon atthe 5′ end of the DNA coding for preproparathyroid hormone. The presenceof the double start codon may cause a production microorganismstransformed with a plasmid containing the cDNA to producepreproparathyroid hormone at an increased rate and in an improved yieldover prior art transformed microorganisms.

[0015] There is further provided by the present invention a plasmid forinsertion into yeast containing DNA coding for parathyroid hormone. In apreferred embodiment, this plasmid is prepared by recloning the plasmidfor insertion in E. coli described above. Moreover, the inventionprovides a yeast transformed by said plasmid for insertion in yeast suchthat the yeast produces and secretes parathyroid hormone. Thus, theinvention provides a method by which parathyroid hormone may be isolatedfrom yeast culture medium. In a preferred embodiment, the transformedyeast is Saccharomyces cerevisiae. In another preferred embodiment, theparathyroid hormone is human parathyroid hormone.

[0016] By use of in vitro mutagenesis, the present invention alsoprovides substitution of one or more amino acids in human parathyroidhormone and peptides having parathyroid hormone agonistic orantagonistic activity. Further, there are provided analogs, fragments,or extensions of the parathyroid hormone (collectively referred to as“derivatives”) which also show agonistic or antagonistic activity.Examples of these peptides have been produced as secretory products inyeast and in E. coli.

[0017] The present invention further provides different leader sequencesand secretion signal sequences that may be used for the production andsecretion of the PTH hormone and/or its derivatives. In at least oneinstance, an alternate leader sequence provides improved production ofthe desired hormone or derivative.

[0018] Additionally, the invention provides a downstream processtechnology for purification of human parathyroid hormone andderivatives. The process involves a purification procedure yeast or E.coli medium or periplasmic solution, and consists principally of cationexchange chromatography followed by two steps of high pressure liquidchromatography. The final product is more than 95 percent pure and canbe submitted directly to N-terminal amino acid sequencing as well asamino acid composition determination.

[0019] Human parathyroid hormone (hPTH) is a key regulator of calciumhomeostasis. The hormone is produced as a 115 amino-acid prepro-peptide.Before secretion the prepro part is cleaved off, yielding the 84 aminoacid mature hormone. Through its action on target cells in bone andkidney tubuli, hPTH increases serum calcium and decreases serumphosphate, while opposite effects are found regarding urinary excretionof calcium and phosphate. At chronically high secretory rates of PTH(hyperparathyroidism) bone resorption supersedes formation. However,prolonged exposure to low/moderate doses of a biologically activePTH-fragment stimulates bone formation and has also been reported to beeffective in the treatment of osteoporosis by inducing an anabolicresponse in bone (Reeve et al. 1980 Br Med J 250, 1340-1344 Slovik etal. 1986 J Bone Min Ros 1, 577). So far studies on intact hPTH have beenhampered by the limited availability and the high price of the hormone.Hence a system for the efficient expression of hPTH in microorganismswould be very advantageous for the further progression of studies onhPTH and its role in bone biology and disease.

[0020] Poly (A)⁺-selected RNA was isolated from human parathyroidadenomas immediately after surgery. The RNA was size-fractionated, cDNAwas prepared and cloned into the PstI site of pBR322 by the GC-tailingmethod. The library was screened by using synthetic oligonucleotides.Sixty-six clones of a total of 34,000 were found to be positive for both5′ and 3′ PTH sequences. The correct identity of four of these cloneswas verified by DNA sequence analysis.

[0021] Employing the promoter and signal sequence of Staphyloccousaureous protein A we have expressed hPTH in Eacherichis coli as asecretory peptide. Immunoreactive PTH was isolated both from growthmedium and periplasmic space. We obtained up to 10 mg/1 hPTH as judgedby reactivity in radioimmunoassay.

[0022] hPTH was expressed in Saccharomyces ceerevisiae after fusing hPTHcDNA to an expression vector coding for the prepro-region of the yeastmating factor α. During the secretion process, the α-factor leadersequence is cleaved off by an endopeptidase specific for a dibasic aminoacid sequence and encoded by the KEX2 gene.

[0023] By hPTH-specific radioimmunoassay a significant amount of hPTHimmunoreactive material was detected in the growth medium, correspondingto about 1 mg hPTH pr 1 medium, of the yeast strain FL200 transformedwith fusion plasmid pαLXPTH. No immunoreactive hPTH was secreted fromcells transformed with the vector pαLX.

[0024] Parallel cultures of the yeast strain FL200 transformed with oneof the three expression plasmide pUCXPTH, pαUXPTH-1 and pαLXPTH withcopy numbers near unity, normal high (˜30) and very high (>50)respectively were grown and both growth medium, a periplasmic fractionand an intracellular soluble fraction were assayed for hPTHimmunoreactive peptides.

[0025] The results show that the intermediate copy number gave thehighest production. The produced PTH was secreted completely to thegrowth medium. The secreted products were concentrated from the growthmedium and analyzed on SDS-PAGE. A distinct band with the same molecularweight as hPTH standard was visible on the gel.

[0026] hPTH immunoreactive material was concentrated from the growthmedium by passage through a S Sepharose Fast flow column and elutedquantitatively. Recombinant hPTH was purified by reverse phase HPLC. Thecolumn was eluted with a linear gradient of acetonitrile/trifluoroaceticacid. A major peak (fractions 32 and 33) with the same retention time asstandard hPTH(1-84) was resolved into two peaks in a second HPLCurification step. The major peak from the 2.HPLC eluted exactly asstandard hPTH(1-84) and co-chromatographed with hPTH(1-84) as onesymmetric peak. SDS-PAGE of the peak fraction showed one bandco-migrating with hPTH standard suggesting that the recombinant PTH wasessentially pure. The recombinant hPTH was subjected to N-terminal aminoacid analysis. We were able to determine unambiguously 45 amino acidsfrom the N-terminal end in the E. coli protein and 19 amino acids in theyeast protein. The sequence was identical to the known sequence of hPTH.The sequence analysis indicated that the recombinant PTH was more than90 percent pure. The recombinant hPTH from E. coli and Saccharomycescerevisiae was fully active in adenylate cyclase assay and also inducedhypercalcemia in rats after injection.

[0027] We have successfully expressed biologoically active intact humanparathyroid hormone as a secretory peptide in Escherichia coli andSaccharomyces cerevisiae, and developed a down-stream purificationtechnology.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 shows all possible variations of the DNA sequence codingfor human preproparathyroid hormone.

[0029]FIG. 2 shows the specific human preproparathyroid hormone DNAcoding sequence of the clone pSShPTH-10.

[0030]FIG. 3 shows a DNA sequence coding for human preproparathyroidhormone and having a double start codon at the 5′ terminal end withflanking sequences in which are shown all possible variations of the DNAwhich may be present on the plasmid of the present invention.

[0031]FIG. 4 shows the specific human preproparathyroid hormone DNAcoding sequence of the clone pSSHPTH-10 with flanking sequences.

[0032]FIG. 5 shows the actual amino acids sequence of the humanpreproparathyroid hormone for which the DNA sequence in close pSShPTH-10codes.

[0033]FIG. 6 shows the sequence of the MFα1-hPTH fusion gene with allpossible combinations of the DNA coding for hPTH.

[0034]FIG. 7 shows the sequence of the MFα1-hPTH fusion gene.

[0035]FIG. 8. Analysis of expression products by SDS-PAGE andimmonoblotting. Saccharomyces cerevisiae transformed with a PTH cDNAcarrying plasmid was grown in liquid culture medium. The secretedproducts were concentrated and analyzed on SDS-PAGE. Panel a shown asilver stained gel with molecular size marker (lane S), hPTH standard(lane P), and concentrated yeast growth medium (lane l). After blottingonto a PVDF membrane, blots were probed with hPTH specific antibodies,one reactive against the aminoterminal part of the hormone (panel b),another reactive against the middle region of the hormone (panel c).Lanes in panel b and c are numbered as in panel a.

[0036]FIG. 9. Purification of recombinant hPTH from the growth medium.

[0037] A: Chromatogram of the 1.HPLC purification

[0038] B: Chromatogram of the 2.HPLC purification of fractions 32 and 33from panel A. The peak of the recombinant hPTH is indicated by black.

[0039] C: 2.HPLC run of 1 ug standard hPTH(1-84)

[0040] D: Co-chromatography of the recombinant PTH pack from panel B and1 ug of standard hPTH (1-84)

[0041] E: Silver staining of SDS-PAGE of the proteins in the hPTH pack

[0042] 1: recombinant hPTH, 1 ug

[0043] 2: hPTH(1-84) (σ), 3 ug (Note HMW Impurities)

[0044]FIG. 10. Construction of PPTH-M13-ΔEA/KQ.

[0045]FIG. 11. Schematic representation of the mutation introduced inthe gene fusion between the yeast α-factor prepro region and the humanparathyroid hormone.

[0046]FIG. 12. SDS PAGE of concentrated yeast growth medium containingmutated and wild type hPTH. Aliquots of concentrated growth medium fromyeast strain BJ1991 transformed with the expression plasmids pαUXPTH-2⁹(lane 2) and pαUXPTH-Q26 (lane 1) were analyzed by 15% PAGE in thepresence of 0.1% SDS, and visualized by silver staining as described inExperimental Protocol. Lane M shows a molecular size marker including ahPTH standard. The latter is marked with an arrow.

[0047]FIG. 13. Purity of purified hPTH (1-84,Q26). Yeast growth mediumfrom yeast strain BJ1991 transformed with the expression plasmidspαUXPTH-Q26 were concentrated and purified by reversed phase HPLC asdescribed in Experimental Protocol. The purity of the recombinanthormone was then analyzed by analytical HPLC (Panel A) and SDS PAGE(Panel B, lane 2). In Panel B the purified hPTH (1-84,Q36) is comparedwith the wild type hormone purified by two runs on HPLC (lane 3). Themolecular weight market in lane M is the same as in FIG. 2. Lane 1 showsa reference PTH produced in E. coli.

[0048]FIG. 14. Two dimensional gelelectrophoretic analysis of hPTH(1-84,Q26). An aliquot of concentrated growth medium from yeast strainBJ1991 transformed with the expression plasmids pαUXPTH-Q26 wasseparated on an acetic acid 15% PAGE. The two main bands (band 1 and 2)migrating close to the hPTH standard were then cut out, equilibratedwith SDS loading buffer and run into a second dimension 15% PAGEcontaining 0.1% SDS in separate lanes in triplicate. This gel wasdivided in three and one part was colored with silver (Panel A), onepart blotted and treated with hPTH N-terminal region specific antibodies(Panel B) and one part blotted and treated with hPTH middle-regionspecific antibodies (Panel C). Lanes 1 and 2 show band 1 and 2, PTHe isa reference hPTH produced in E. coli, PTH_(c) is a commercial hPTHreference. Lane S shows a molecular weight standard.

[0049]FIG. 15. Biological activity of hPTH (1-84,Q26). Recombinant hPTH(1-84,Q26) (▪) was purified on HPLC and assayed for biological activityin a hormone-sensitive osteoblast adenylate cyclase (AC) assay asdescribed in Materials and Methods. The experiments were carried out intriplicate determinations. hPTH (1-84) from Sigma (O) and recombinantyeast hPTH (1-84) (▴) were used as references.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] As indicated above, the present invention is directed to aplasmid for insertion in E. coli containing DNA coding for humanpreproparathyroid hormone. The invention is also directed to theresulting transformed E. coli.

[0051] The invention further is directed to a plasmid for insertion intoyeast which contains DNA coding for parathyroid hormone and which isderived from the plasmid for insertion into E. coli. Finally, theinvention is directed to a transformed yeast from which parathyroidhormone may be recovered.

[0052] The invention further provides methods of producing and isolatingthe plasmids and transformed microorganisms. Poly(A) selected RNA wasisolated from human parathyroid adenomas collected immediately aftersurgery. The poly(A) RNA was enriched for correct size mRNA byultracentrifugation through sucrose gradients. Preproparathyroid hormoneof correct molecular weight was translated in vitro from this sizefractionated poly(A) RNA as judged by sodium dodecylsulphatepolyacrylamide gel electrophoreses after immuno precipitation withantiparathyroid antiserum. The specific messenger RNA for the human PTHwas used as template for complementary DNA synthesis using oligo d(T)18as a primer and avian myoblastosis virus reverse transcriptase. Afterremoval of the RNA templates by alkali hydrolysis, the second strandcomplementary DNA was synthesized by incubating the purified firststrand DNA in the presence of the Klenow fragment of E. coli DNApolymerase I. The double stranded comlementary DNA was made blunt endedby the action of Aspergillus oryzae single strand specific endonucleaseS1 and complementary DNA longer than 500 base pairs was isolated afterneutral sucrose gradient centrifugation. Approximately 20 bases longd(C)-tail protrusions were enyzmatically added to the 3 ends of thecDNA. This modified complementary DNA was annealed to restrictionenconuclease PstI cleaved and d(G)-tailed vector pBR322. Resultingrecombinant plasmid DNA's were transformed into E. coli KI2 BJ 5183.Positive transformants were analyzed for by colony hybridization usingtwo different synthetic deoxyribooligonucleotides which covered theN-terminal coding region as well as the 3′ non-coding part of thehormone mRNA sequence, respectively. Six out of 66 clones that werepositive for both probes were submitted for detailed analysis byrestriction endonuclease mapping showing that they all were identicalexcept for some size heterogenity at the regions flanking the startcodon and the XbaI site 3′ for the stop codon. One clone, pSShPTH-10,was subjected to DNA sequence analysis revealing a 432 nucleotide longhuman parathyroid hormone complementary DNA sequence inserted in thePstI site of pBR 322. The entire cDNA sequence was found to be identicalto the sequence previously described by Hendy, et al., supra, except fora 5 base pair deletion in front of the start codon.

[0053]FIG. 2 shows the human preproparathyroid hormone DNA sequence ofpSShPTH-10. This may be compared with FIG. 1, which shows all possiblevariations of the DNA sequence for human preparathyroid hormone withoutthe 5′ double start codon. FIG. 3 shows the DNA sequence of the clone ofthe present invention with the flanking sequences. In a preferredembodiment, the plasmid for insertion in E. coli coding for humanpreproparathyroid hormone is pSShPTH-10, the DNA sequence of which,including the flanking sequence, is shown in FIG. 4.

[0054] The invention further provides a plasmid for insertion into yeastcontaining DNA coding for parathyroid hormone. The parathyroid hormonemay be human or animal parathyroid hormone, for example pig or bovineparathyroid hormone. The plasmid for insertion in yeast of the presentinvention may be recloned from plasmids containing DNA coding for humanor animal parathyroid hormone. In a preferred embodiment, the plasmidfor insertion in yeast contains DNA coding for human parathyroidhormone. As shown in the following examples, the hTPH sequence frompSShPTH-10 has been recloned and inserted in designed vectors forexpression in Saccharomyces cerevisiae.

[0055] pSShPTH-10 was digested to form a 288 bp BglII-XbaI fragment.This fragment was then subcloned into pUC19 between the BamHI and XbaIsites. The subclone was then digested with Dpn I, and the largestresulting fragment was isolated. The said fragment was then digestedwith SalI.

[0056] The plasmid pSSαLX5-hPTH1 that in yeast MAT cells leads to theexpression and secretion of PTH was constructed in three stages:

[0057] 1. Construction of the yeast shuttle vector pL4 (which replicatesin both E. coli and Saccharomyces cerevisiae).

[0058] 2. Cloning of a DNA fragment containing the yeast matingpheromone MFα1 gene and its insertion into the yeast shuttle vector tomake the pαLX5 vector.

[0059] 3. Insertion of a DNA fragment from the coding region of the hPTHgene of pSShPTH-10 into pαLX5 in reading frame with the prepro part ofthe MF 1 gene, thereby producing the vector pSSαLX5-hPTH1.

[0060] The shuttle vector pL4 was constructed by inserting into pJDB207,an EcoRI-AvaII fragment containing the ADHI promoter isolated fromPADHO40. A SphI fragment was then deleted, resulting in a plasmid pALX1.The PstI site in the B-lactamase gene was deleted and the plasmid waspartially digested with PvuI and BglI and ligated to a PvuI BglIfragment of pUC8, to form pALX2. After a further oligonucleotideinsertion, the plasmid was digested with HindIII and religated to formpALX4.

[0061] Total yeast DNA from the Y288C strain was digested with EcoRI,and the 1.6-1.8 kb fragments isolated. These were ligated toEcoRI-cleaved pBR322, and E. coli was transformed. The clones werescreened for MFα1 inserts by oligonucleotide hybridization. The DNAselected thereby was then used to transform E. coli. The resultingplasmid pMFα1-1 was digested with EcoRI, made blunt ended by Klenowenzyme, and then digested with BglII. The MFα1 fragment was isolated,and ligated to pL5 (digested with BamHI, made blunt ended with Klenowenzyme, and digested with BglII) to yield pαLX5.

[0062] In order to insert the human PTH CDNA fragment into pαLX5, thepαLX5 was digested with HindIII, creating sticky ends and the site wasmade blunt ended with the DNA polymerase I Klenow fragment and dNTP. ThepαLX5 was then digested with SalI to create a sticky ended DNAcomplementary to the SalI digested human PTH fragment described above.

[0063] The SalI digested human PTH fragment was then inserted into theSAlI digested pαLX5. The resulting plasmid pSSαLX5-PTH was then insertedinto yeast, thereby transforming yeast so that the yeast produces andsecretes intact human parathyroid hormone. In a preferred embodiment,the transformed yeast is Saccharomyces cerevisiae.

[0064] As explained above, the invention provides alternate leadersequences which may be used for the production of parathyroid hormone orderivatives thereof, as taught by the present invention. The method setforth above discloses the use of the α-factor leader sequence. However,other sequences may be used, at least one of which has been shown toprocess PTH with greater efficiency than does the entire α-factor leadersequence. It has been discovered that the deletion from the α-factorleader of a 12-base sequence which comprises the yeast STE13 recognitionsite produces a more efficient production mechanism for PTH and/or itsderivatives. PSSαUXPTH-ΔEA contains the α-factor hPTH fusion gene placedbetween the α-factor promoter and terminator, in which the regionencoding the Glu-Ala-Glu-Ala recognition sequence of the yeast STE13aminopeptidase has been deleted. As another example of an alternativeleader sequence, a leader sequence comprised of only the first nineteenamino acids of the α-factor is also used in the method of the presentinvention.

[0065] Also shown is an example of site specific mutagenesis changingthe codon for the amino acid 26 in the PTH gene, thereby transforming alysine-codon (K) to glutamine-codon (Q) using the Muta-Gene™ in vitromutagenesis kit from Bio-Rad. For this purpose, the plasmidpαPTH-M13-ΔEA was used to transform the E. coli strain CJ236. Auracil-containing single-stranded DNA which was prepared from the phagewas annealed to a synthetic oligonucleotide, and second strain synthesiswas carried out with T4 DNA polymerase and ligation with T4 DNA ligase.The heteroduplex DNA was transformed into the E. coli strain MV1190 tobe repaired into a homoduplex by removal of uracil incorporated in theparental strand. Positive clones were verified by DNA sequencing and oneof these was called pαPTH-M13-ΔEA/KQ. Finally, the entire expressioncassette between a BamHI and a filled-in EcoRI site was isolated fromthis vector construction and inserted into the BamHI and PvuII site ofthe yeast shuttle vector YEp24 and this final expression plasmid wasdesignated pSSαUXPTH-ΔEA/KQ.

[0066] A point mutation was introduced in the gene encoding the humanparathyroid hormone leading to a change of the 26th amino acid fromLysine (K26) to Glutamine (Q26). When this gene was expressed andsecreted in Saccharomyces cerevisiae using the α-factor fusion system,the full length hormone was found in the growth medium with nodegradation products present. This contrasts the situation when the wildtype gene is expressed in the same system. Then the major product is ahormone fragment hPTH(27-84), and only up to 20% of the immunoreactivesecreted material is hPTH(1-84). The yield after a two step purificationof the degradation resistant hormone was 5-10 fold higher than what wasobtained with the wild type hormone. The secreted hPTH(1-84,Q26) hadcorrect size, full immunological. reactivity with two different hPTHspecific antibodies and correct N-terminal amino acid sequence.Furthermore, the introduced mutation had no effect on the biologicalactivity of the hormone as judged from its action in a hormone-sensitiveosteoblast adenylate cyclase assay.

[0067] Human parathyroid hormone (hPTH) is one of the key calciumregulating hormones in the body. The hormone is, produced in theparathyroid gland as a 115 amino acid prepro-peptide that is processedduring secretion to an 84 amino acid mature hormone.^(1/) It actsprimarily on kidney and bone cells, stimulating calcium back resorptionand calcium mobilization, respectively.^(2-4/) The hormone seems toexhibit differential catabolic as well as anabolic effects and itsoverall physiological action is probably to generate a positive calciumbalance and enhance bone formation. The area of potential utilityincludes possible use in treatment of postmenopausal osteoporosis aswell as in prevention of postpartum hypocalcaemia in cows. Sufficientsupplies of authentic recombinant hPTH are of considerable interest toevaluate such applications.

[0068] hPTH is an easily degraded polypeptide. Already in theparathyroid gland large amounts of carboxyl-terminal PTH fragments aregenerated.^(1/) Structural studies have suggested that hPTH may containtwo domains with the easily cleaved region placed in a connecting stalkbetween these domains.^(5/) Not surprisingly therefore, degradation ofhPTH has been a major problem when the hormone is expressed inheterologous organisms. In E. coli low expression levels combined withdegraded hormone peptides of short half-life were observed.^(6-8/) Themost successful expression system for hPTH so far is Saccharomycescerevisiae where the hormone is expressed as a secretory peptide.^(9/)By that approach we were able to obtain significant amounts of authentichPTH(1-84) with full biological activity. But even if conditions werefound which eliminated proteolytic attacks at some sites in the putativestalk region of the hormone, a significant fraction of the secretedpeptides was still cleaved after a pair of basic amino acids found inthe hPTH sequence reducing the yield of full length peptide hormone. Thecleavage site resembles that recognized by the yscF protease (the KEX2gene product).^(10.11/) We reasoned that the elimination of the putativeyscF cleavage in hPTH could lead to a significant gain in the yield ofundegraded hPTH secreted from yeast. In the present report we describethe removal of the putative yscF cleavage sites by in vitro mutagenesisof the hPTH coding region. When the amino acid at position 26 in hPTHwas changed from Lysine (K26) to Glutamine (Q26), the major degradationproduct hPTH(27-84) previously observed disappeared in the growth mediumand the yield of full-length hormone increased 5- to 10-fold. Thesecreted degradation resistant hPTH)1-84, Q26) had correct size, fullimmunological reactivity with two different hPTH specific antibodies andcorrect N-terminal amino acid sequence. Furthermore, the introducedmutation had no effect on the biological activity of the hormone asjudged from its action in a hormone-sensitive osteoblast adenylatecyclase assay.

[0069] The Saccharomyces cerevisiae strain used for the hPTH expressionwas BJ1991 (a, trpl, ura3-52, leu2, prb1-1122, pep4-3). Yeast cells weretransformed by the lithium method^(12/), and transformants grown at 30°C. in YNBGC medium (0.67 percent yeast nitrogen base, 2 percent glucose,1 percent casamino acids (Difco).

[0070] The paUXPTH-2 plasmid used as a reference for expression ofauthentic hPTH(1-84) is described.^(9/) In order to change the codon 26in the hPTH gene from AAG (Lysine) to CAG (Glutamine), an a-factor hPTHgene fusion subcloned in M13 mp19 (designated M13PTH-3 in ^(9/)) wasmodified by in vitro mutagenesis using the “Muta-gene™ in vitromutagenesis kit” (Bio-Rad) based on the method of Kunkel et al.^(13/).The mutagenizing oligonucleotide had the sequence5′-GGCTGCGTCAGAAGCTGC-3′ where all nucleotides except the ninth arecomplementary to the actual hPTH sequence. Positive clones were verifiedby DNA sequencing.^(14/) One of those were picked and called M13PTH-Q26.The entire expression cassette between a BamHI and a filled in EcoRIsite was finally isolated from M13PTH-Q26 and inserted between the BamHIand PvuII site of the yeast shuttle vector YEp24.^(15/) This expressionplasmid was designated paUXPTH-Q26. The translation product from thehPTH gene between amino acid 25 and 27 should now change fromArg-Lys-Lys to Arg-Gln-Lys.

[0071] Radioimmunoassay of hPTH in yeast culture media was carried outas described.^(9/,16/). For electrophoretic analysis, yeast culturemedia were concentrated as previously described^(9/), and separated on a15 percent polyacrylamide gel in the presence of SDS^(17/), and eitherstained with silver^(18/) or further analyzed by protein blotting usingImmobilon PVDF Transfer Membranes (Millipore) and the buffers of Towbinet al.^(19/) Reference hPTH(1-84) was purchased from PeninsulaLaboratories (USA). Protein blots were visualized as described.^(9/)

[0072] The concentrated medium from the Sepharose S column was subjectedto further purification by reversed phase HPLC using a Vydac proteinpeptide C18 column (The Separation Group, Hesperia, Calif., USA). Thecolumn was eluted with a linear gradient of acetonitrile/0.1 percenttrifluoroacetic acid.

[0073] Proteins to be sequenced was purified either by HPLC as describedabove or by SDS polyacrylamide gelelectrophoresis followed by blottingonto polyvinylidene difluoride membranes.^(20/) Automated Edmandegradation was performed on a 477A Protein Sequencer with an on-line120A phenylthiohydantoin amino acid analyzer from Applied Biosystems(Foster City, Calif., USA). All reagents were obtained from AppliedBiosystems.

[0074] The adenylate cyclase stimulating activity of the recombinanthPTH was assayed as previously described^(9,21,22/) hPTH(1-84) fromSigma was used as, reference.

[0075] Different strategies could be envisaged to avoid the degradationof parathyroid hormone during expression in heterologous organisms. Onerecently reported strategy is to express intracellularly in E. coli acro-lacZ-hPTH fusion protein that subsequently is cleaved by strong acidto give proline-substituted hPTH.^(23/) However, since secretion of thehormone in yeast seems to be a more efficient way of producing acorrectly processed hormone, and also is superior with respect todownstream processing, we rather adopted a strategy to improve thissystem. Only one major cleavage site is used during secretion in yeastwhen the cells are grown under proper conditions: after a pair of basicamino acids in position 25 and 26 in the hPTH sequence. This cleavagesite resembles that recognized by the yscF protease (the KEX2 geneproduct). We reasoned that a substitution of a glutamine for the lysine26, as illustrated in FIG. 11, ought to be a structurally conservativechange that should exclude the hormone as a substrate for the yscFprotease.

[0076] The yeast strain BJ1991 was transformed with the plasmidspaUXPTH-Q26 containing the mutated hPTH coding region. One transformantwas grown in YNBGC medium lacking uracil and the cell free medium wasconcentrated and analyzed in different gel systems. FIG. 12 shows asilver-stained SDS polyacrylamide gel where concentrated medium frompaUXPTH-Q26 transformed cells (mutated hPTH, lane 1) is compared withthat from paUXPTH-2 transformed cells (wild type hPTH, lane 2). In thelatter case the strongest band has a molecular mass lower than thestandard hPTH, and previous microsequencing has shown that itcorresponds to the hormone fragment hPTH(27-84). In the lane with themutated product (lane 1), this band is absent showing that the cleavagebetween amino acid 26 and 27 has been totally eliminated as a result ofthe mutation. Now the major product is a polypeptide that migrates closeto the full length hPTH standard. Consistently, this band had amigration slightly faster than the standard in an anionic gel system anda migration slightly slower than the standard in a cationic gel systemin accordance with the single charge difference between the mutated (onepositive charge less) and the wild type hormone. In addition to the mainproduct a few weaker bands were present of apparently higher molecularmass which might be O-glycosylated forms of the hormone.

[0077] This hPTH(1-84,Q26) candidate was further analyzed by twodimensional gel electrophoresis and protein blotting. In the firstdimension acetic acid/urea gel a simple pattern with mainly two bandswas found. These were cut out and run on a second dimension SDSpolyacrylamide gel. The silver stained second dimension gel as well astwo protein blots probed with different PTH antibodies, are shown inFIG. 14. The hPTH(1-84,Q26) candidate migrating closest to the hPTHstandard in both dimensions, reacted with two hPTH specific antibodiesraised against N-terminal region and the middle/C-terminal region of thehPTH respectively.

[0078] The nature of the hPTH(1-84,Q26) candidate was finally confirmedby N-terminal amino acid sequencing, both directly oh the polypeptideband after blotting onto a PVDF membrane filter, and after purificationon reversed phase HPLC. Correct amino-terminal sequence was found inboth cases. Furthermore, the expected change from lysine to glutamine inposition 26 was confirmed by sequencing through this position.

[0079] Since the elimination of the internal cleavage of the secretedhPTH leads to fewer polypeptides with similar properties in the growthmedium, this form of the hormone could also be isolated by a simplifiedpurification procedure. Already in the first concentration step using aSepharose S column, a certain purification is achieved. All hPTHimmunoreactive material is retained, but some high molecular weightmaterial is removed in the pH6 wash of the Sepharose S column. Thisfirst concentrated eluate already contained more than 80 percenthPTH(1-84, Q26). Then, a single run on a reversed phase HPLC C18 column,was enough to give near homogeneous hPTH(1-84, Q26). The purity waschecked both by SDS polyacrylamide gelelectrophoresis and sensitivesilver-staining, and by analytical HPLC as illustrated in FIG. 13A. Asingle peak is found in the chromatogram (FIG. 13A), and a single bandwith only a trace of a closely migrating hPTH band (probably anO-glycosylated form of the hormone) could be seen in the SDSpolyacrylamide gel (FIG. 13B). When the yield of pure full lengthmutated hormone was compared with that of the wild type, 5 to 10 foldhigher yields were usually achieved. This is consistent with ourprevious estimate of the fraction of full length hormone (up to 20percent) obtained when the wild type is expressed.^(9/)

[0080] The biological activity of the secreted hPTH(1-84,′ Q26) wastested in a hormone-sensitive osteoblast adenylate cyclaseassay.^(9,21,22/) The purified hPTH(1-84, Q26) was analyzed for itsability to stimulate the adenylate cyclase activity of OMR 106osteosarcoma cells above the basal level. The quantitative analysisshown in FIG. 15, clearly demonstrates that hPTH(1-84, Q26) has astimulatory effect comparable to that of a commercial hPTH control. Thestimulation curve practically coincides with that of purifiedrecombinant wild type hPTH(1-84). Consequently, no difference inbiological activity could be detected between the wild type hormone andthe degradation resistant mutated hormone.

[0081] We have shown that the easily degraded human parathyroid hormonecan be expressed in a correctly processed and intact form, inSaccharomyces cerevisiae after the introduction of a single,structurally conservative mutation in the 26th amino acid of thehormone. The increase in final yield of pure full length hormone is 5-to 10-fold compared to what is obtained with wild type hormone expressedin the same system. The mutation also simplifies the downstreampurification of the hormone. A concentration step followed by a singleHPLC run was enough to give near homogeneous recombinant hormone.

[0082] We have previously described conditions of growth that eliminatessecondary cleavages in the protease sensitive “stalk” region of thehormone ^(9/). Here we describe how the final dibasic cleavage site canbe eliminated. After introduction of the mutation, a form of the hormoneis produced that totally resists the frequent cleavage found in the wildtype hormone after the Arg25-Lys26 motif. The possible internal cleavageat putative dibasic amino acids is one of the severe drawbacks of theα-factor secretion system. To our knowledge this is the first reportedcase where this problem has been successfully overcome.

[0083] Previous reports have shown that the biological activity of thehormone resides in the first third of the molecule in a minimumstructure comprised of amino acids 1-27. Furthermore, the triple basicamino acid motif from position 25-27 seems to be conserved between thebovines^(25/), porcine^(26/) and human hormone^(27/). It was thereforenot obvious that the introduction of a mutation in position 26 would notdestroy the biological activity of hPTH. However, no difference betweenthe recombinant hPTH products could be detected in the adenylate cyclaseassay, showing that the introduced mutation does not affect thebiological activity of the hormone.

[0084] hPTH is a multifunctional hormone with many potential uses, forexample in diagnostics and as a drug in veterinary medicine. A fragmentof hPTH together with 1,25(OH)₂ vitamin D₃ has also been reported toinduce bone formation in humans ^(27, 28/), and one of the major areasof potential use of a recombinant hPTH is therefore in the treatment ofosteoporosis. To evaluate such applications, sufficient supplies ofrecombinant hPTH are essential. In the present report we have describedwhat we believe is the most efficient way of producing full lengthbiologically active parathyroid hormone so far.

[0085] Moreover, the method of the present invention may be used toproduce parathyroid hormone derivatives having parathyroid hormoneagonistic or antagonistic activity. These derivatives include hormoneanalogs, such as the example described above in which the lysine atposition 26 is substituted with glutamine, or may be fragments orextensions of the hormone, i.e., polypeptides having parathyroid hormoneagonist or antagonist activity which are respectively shorter or longerthan the hormone itself. Parathyroid hormone agonistic effect in thisconnection will be demonstrated by activation of adenylyl cyclase inbone cells and kidney cells. The in vivo effects of such activity mimicthe effects of native parathyroid hormone with respect to plasma calciumconcentration alterations as well as the well known hormonal actions oncalcium and phosphate re-absorption and excretion in the kidney.Furthermore, the PTH derivatives of the present invention having agonistactivity shall also have the capacity to reduce the alkaline phosphataseactivity of certain osteoblast cell lines, and stimulate ornithinedecarboxylase activity bone cells (UMR 106 cells) or chicken condrocytesand stimulate DNA synthesis in chicken condrocytes. Moreover, thederivatives shall have the capability of blocking the action ofparathyroid hormone itself or of any of the other agonist derivatives.

[0086] The invention also provides alternate secretion signal sequencesfor the secretion of the PTH hormone or its derivatives from yeast. Asdisclosed above, parts of the MFα1 gene may be inserted into the plasmidof the present invention to cause the yeast to secrete the intact PTHhormone or derivatives. However, other signal sequences will alsofunction in the methods of the present invention. The process of proteinsecretion requires the protein to bear an amino-terminal signal peptidefor correct intracellular trafficking, the sequence of which is termed“signal sequence”. Two classes of signal sequences will function in theplasmids of the present invention, and will cause secretion of the PTHhormone or derivative from yeast: “optimalized consensus signalsequences” and other functional signal sequences. An “optimalizedconsensus signal sequence” is any amino-terminal amino acid sequencethat is composed of the following three parts:

[0087] 1. An amino-terminal positively charged region. The size of thisregion may vary from 1-20 amino acids. The only specific characteristicis a positive charge at physiological pH conferred by the presence ofone to three basic amino acids (Lys or Arg).

[0088] 2. A hydrophobic core region. The size of this region may varyfrom 7-20 amino acids, and it is predominantly composed of hydrophobicamino acids (Phe, Ile, Leu, Met, Val, Tyr, or Trp).

[0089] 3. A polar COOH-terminal region composed of five amino acids(from position -5 to -1 relative to the cleavage site) that defines thecleavage site. The specific character of this region is that the aminoacid in position -1 must be a small neutral amino acid (Ala, Ser, Gly,Cys, Thr, or Pro), and that the amino acid in position -3 must be eithera hydrophobic amino acid (Phe, Ile, Leu, Met, Val) or a small neutralamino acid (Ala, Seri Gly, Cys, Thr, or Pro).

[0090] See von Heijne, G. (1983) “Patterns of Amino Acids nearSignal-Sequence Cleavage Sites.” Eur. J. Biochem. 133, 17-21, and vonHeijne, G. (1985) “Signal sequences. The limits of variation.” J. Mol.Biol. 184, 99-105. However, Kaiser, C. A., Preuss, D., Grisafi, P., andBotstein, D. (1987) “Many Random Sequences Functionally Replace theSecretion Signal Sequence of Yeast Invertase.” Science 235, 312-217,found the specificity with which signal sequences were recognized inyeast to be low and that any amino-terminal peptide with ahydrophobicity above some threshold value would function. Therefore,“functional signal sequence” is defined as any amino-terminal amino acidsequence that can direct secretion in yeast even if it does not fit allthe criteria of an optimal signal sequence.

[0091] Specific examples of signal sequences functional in yeast thatconform to the description of an optimal signal sequence are:

[0092] 1. Met,Lys,Ala,Lys-Leu,Leu,Val,Leu,Leu,Thr,A la,Phe-Val,Ala,Thr,Asp,Ala (Jabbar, M. A., and Nayak, D. P. (1987) “SignalProcessing, Glycosylation, and Secretion of Mutant Hemagglutinins of aHuman Influenza Virus by Saccharomyces cerevisiae.” Molec. Cell. Biol.7, 1476-1485.) from a human influenza virus hemagglutinin.

[0093] 2. Met,Arg,Ser-Leu,Leu,Ile,Leu,Val,Leu,Cys,P he,Leu,Pro-Leu,Ala,Ala,Leu,Gly (Jigami, Y., Muraki, M., Harada, N., andTanaka, H. (1986) “Expression of synthetic human-lysozyme gene inSaccharomyces cerevisiae: use of a synthetic chicken-lysozyme signalsequence for secretion and processing.” Gene 43, 273-279.) from chickenlysozyme.

[0094] 3. Met,Arg,Phe,Pro,Ser-Ile,Phe,Thr,Ala,Val,L eu,Phe,Ala,Ala-Ser,Ser,Ala,Leu,Ala (Ernst, J. F. (1988) “EfficientSecretion and Processing of Heterologous Proteins in Saccharomycescerevisiae is mediated solely by the Pre-Segment of α-factor Precursor.”DNA 7, 355-360. Kurjan, J. and Herskowitz, I. (1982) “Structure of aYeast Pheromone Gene (MFa): “A putative α-factor Precursor contains fourTandem Copies of Mature α-factor”. Cell 30, 933-934.) from yeastα-factor precursor.

[0095] A specific example of signal sequences functional in yeast thatconforms to the description of a functional signal sequence isMet,Asn,Ile,Phe,Tyr,Ile,Phe,Leu,Phe,Leu,Ser,Phe,Val-Gln, Gly,Thr,Arg,Gly. Baldari, C., Marray, J. A. H., Ghiara, P., Cesareni, G.,and Caleotti, C. L. (1987) “A novel leader peptide which allowsefficient secretion of a fragment of human interleukin 1B inSaccharomyces cerevisiae.” EMBO J. 6. 229-234. from Klyveromyces laciskiller toxin.

[0096] Finally, the invention provides three different steps which takentogether, represent an effective and convenient procedure forpurification of human recombinant parathyroid hormone (PTH). A cationexchange chromatography using S-Sepharose column as described in thetext, washed at pH 6 and eluted at pH 8.5. The immunoreactivity of theintact PTH migrates within the peak.

[0097]FIG. 9 shows high performance liquid chromatography (HPLC) of hPTHwhich was eluted with trifluoraecetic acid and a linear gradient ofacetonitril of 35-60%. The position of intact hPTH is indicated in thesecond HPLC step the acetonitril gradient has been changed to 40-45% andintact hPTH elutes as one symmetrical peak.

[0098] Although the methods of making the invention disclosed herein areshown in detail, these methods are presented to illustrate theinvention, and the invention is not limited thereto. The methods may beapplied to a variety of other plasmids containing DNA coding for humanor animal PTH to produce the plasmids for insertion in yeast of thepresent invention.

[0099] The plasmids of the present invention and transformedmicroorganisms were produced as set forth in the following examples.

EXAMPLE 1 Isolation of mRNA and Synthesis of Complementary DNA (cDNA) ofHuman Parathyroid Hormone

[0100] Starting material for the invention was parathyroid adenomasobtained from patients by surgery. The parathyroid tissue was placed ondry ice directly after removal and transported to a laboratory forpreparation of RNA. The frozen tissue was homogenized with an ultraTurax homogenizer in the presence of 4 M Guanidinium thiocyanate and theRNA content was recovered by serial ethanol precipitations as describedby Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J.,18 Biochemistry 5294-5299 (1979). The RNA preparation was applied tooligo d(T) cellulose affinity chromatography column in order to enrichfor poly(A) containing mRNA. The poly(A) rich RNA was further enrichedfor parathyroid hormone (PTH) mRNA sized RNA by ultracentrifugationthrough a 15-30% linear sucrose gradient. The resulting gradient wasdivided into 25 fractions and every third fraction was assayed for PTHmRNA content by in vitro translation followed by immunoprecipitationwith anti PTH antiserum (Gautvik, K. M., Gautvik, V. T. and Halvorsen,J. F., Scand. J. Clin. Lab. Invest. 43, 553-564 (1983)) andSDS-polyacrylamide gel electrophoresis (Laemmeli, U.K., 227 Nature 680(1970)). The RNA from the fractions containing translatable PTH mRNA wasrecovered by ethanol precipitation. This RNA, enriched for PTH mRNA, wasused as a template for cDNA synthesis using oligo d(T)18 as a primer andavian myoblastosis virus reverse transcriptase for catalysis of thereaction (Maniatis, T., Fritsch, E. F. and Sambrook, J., MolecularCloning pp. 230-243 (1982)). After first strand synthesis, the RNAtemplates were removed by alkali hydrolysis. The second strand cDNA wassynthesized by incubating the purified first strand cDNA in the presenceof the Klenow fragment of E. coli DNA polymerase I (Maniatis, supra).This in vitro synthesized double stranded cDNA was made blunt ended bythe action of Aspergillus oryzae single strand specific endonuclease S1(Maniatis, supra). The blunt ended double stranded cDNA was sizefractionated over a 15-30% neutral sucrose gradient. The sizedistribution of each fraction was estimated by agarose gelelectrophoresis together with known DNA fragment markers. Fractionscontaining cDNA larger than approximately 500 base pairs were pooled andthe cDNA content was collected by ethanol precipitation.

EXAMPLE 2 Cloning of cDNA PTH in Plasmid pBR 322 and Transformation ofE. coli K12 BJ5183

[0101] An approximate 20 base long d(C)-tail protrusion wasenzymatically added to the 3′ ends of the cDNA by the action of terminaldeoxynucleotidyl transferase (Maniatis, supra). The d(C)-tailed cDNA wasannealed to restriction endonuclease Pst I cleaved and d(G)-tailedvector pBR322 and the resulting recombinant plasmid DNA's weretransformed into E. coli K12 BJ 5183 cells which were made competent bythe method of Hanahan, D., 166 J. Mol. Biol. 166, 557-580 (1983). Atotal of 33,000 transformants were analyzed for PTH cDNA content bycolony hybridization (Hanahan, D. and Meselson, Gene 10, 63 (1980)).

[0102] Two to three thousand transformants were plated directly on each82 mm diameter nitrocellulose filter, placed on top of rich medium agarplates containing tetracycline, and incubated at 37 degrees Centigradeuntil approximately 0.1 mm diameter colonies appeared. Duplicatereplicas of each filter was obtained by serial pressing of two newfilters against the original filter. The replica filters were placed ontop of new tetracycline containing agar plates and incubated at 37degrees Centigrade until approximately 0.5 mm diameter coloniesappeared. The master filter with bacterial colonies was kept at 4degrees Centigrade placed on top of the agar plate and the duplicatereplica filters were removed from the agar plates and submitted to thefollowing colony hybridization procedure.

EXAMPLE 3 Characterization of Bacterial Clones Containing RecombinantcDNA PTH and of the DNA Sequence of Clone pSSHPTH-10

[0103] The cells in the respective colonies were disrupted in situ withalkali and sodium chloride leaving the DNA content of each bacterialclone exposed. The procedure allows the DNA to bind to the filter afterwhich it was neutralized with Tris-buffer and dried at 80 degreesCentigrade. The majority of cell debris was removed by a 65 degreeCentigrade wash with the detergent sodium dodecylsulphate (SDS) andsodium chloride leaving the DNA bound to the filters at the position ofthe former bacterial colonies. The filters were presoaked in 6×SSC (0.9M NaCl, 0.09M Na-citrate), 1×Denhart's solution (0.1 g/ml FIcoll, 0.1g/ml polyvinyl pyrrolidone, 0.1 g/ml bovine serum albumin), 100 g/mlherring sperm DNA, 0.5% SDS and 0.05% sodium pyrophosphate for 2 hoursat 37 degrees Centigrade (Woods, D. E. 6 Focus Vol. No. 3. (1984)).

[0104] The hybridization was carried out at 42 degrees Centigrade for 18hours in a hybridization solution (6×SSC, 1×Denhart's solution, 20 g/mltRNA and 0.05% sodium pyrophophate) supplemented with 32P-labelled DNAprobe. (Woods supra).

[0105] The DNA used as a hybridization probe was one of two differentsynthetic deoxyribo oligonucleotides of which the sequences were deducedfrom the published human PTH cDNA sequence of Hendy, supra. The firstprobe was a 24-mer oligonucleotide originating from the start codonregion of the human preproPTH coding sequence having a nucleotidesequence reading TACTATGGACGTTTTCTGTACCGA. The second oligonucleotidewas a 24-mer spanning over a cleavage site for the restrictionendonuclease XbaI located 31 nucleotides downstream of the terminationcodon and consisted of the nucleotide sequence CTCAAGACGAGATCTGTCACATCC.

[0106] Labelling was carried out by transfer of 32 P from 32 P-γ-ATP tothe 5′ end of the oligonucleotides by the action of polynucleotidekinase (Maxam, A. M. and Gilbert, W., 65 Methods Enzymol., 499 (1980)).

[0107] The hybridized filters were washed, in 6×SSC, 0.05% sodiumpyrophosphate at 42 degrees Centigrade prior to autoradiography.Sixty-six clones were found to be positive for both probes as judgedfrom hybridization to both copies of the duplicate replica filters. Allthose were picked from the original filters with the stored cDNA libraryand amplified for indefinitive storage at −70 degrees Centigrade. Six ofthese were chosen for plasmid preparation and a more detailed analysisby restriction endonuclease mapping, showing that all were identicalexcept for some size heterogenity at the regions flanking the startcodon and Xba I site, respectively.

EXAMPLE 4 Clone pSShPTH-10

[0108] One clone, pSShPTH-10, was subjected to DNA sequence analysisaccording to the method of Maxam and Gilbert, supra. This clone consistsof a 432 base pair long PTH cDNA sequence inserted in the Pst I site ofpBR322 having 27 G/C base pairs at the 5′ end and 17 G/C base pairs atthe 3′ end. The complete DNA sequence of the cDNA insert of pSSHPTH-10is shown in FIG. 4. It is identical to the sequence of Hendy, et al.,supra except for a five base pair deletion right in front of the startcodon, changing the published (Hendy, supra) start-stop (ATGTGAAG)signal (deletion is underlined) preceding the used start codon (ATG) toa double start signal (ATGATG).

EXAMPLE 5 Construction of the Yeast Shuttle Vector PL4

[0109] Before the hPTH-yeast-expression project was initiated, a familyof general yeast expression vectors were developed. One of these, pL4,later was used to make pSS LX5-hPTH1, as described below:

[0110] The plasmid pJDB207, constructed by Beggs, J. D., “Multiple-copyyeast plasmid vectors,” Von Wettstein, D., Friis, J., Kielland-Brandt,M. and Stenderup, A. (Eds) Molecular Genetics in Yeast (1981), AlfredBenzon Symposium Vol. 16, 383-390, was chosen as the basis for thegeneral expression vectors. It contains an EcoRI fragment of the yeast 2micron DNA inserted into the pBR322 derivative pAT153. It also containsthe yeast LEU2 gene. The copy number of pJDB207 in yeast cir⁺ cells isvery high relative to that of other plasmids and it is unusually stableafter non-selective growth in a cir⁺ strain, Parent, S. A., Fenimore, C.M., and Bostian, K. A. “Vector Systems for the Expression, Analysis andCloning of DNA Sequences in S. cerevisiae”. 1 Yeast 83-138 (1985);Erhart, E. and Hollenberg, C. P., “The Presence of a Defective LEV2 Geneon 2 Micron DNA Recombinant Plasmids of Saccharomyces cerevisiae isResponsible for Curing and High Copy Number,” 156 J. Bacteriol. 625-635(1983). These properties are related to a partial defective promoter inthe selective marker gene LEU2 (often named LEU2d, d for defective),Erhart et al., supra, which is not changed in the following constructs.

[0111] A 1260 base pair EcoRI-AvaII fragment containing the ADHIpromoter was isolated from the plasmid pADH040. After a fill in reactionwith the Klenow fragment of DNA polymerase I and all four dNTPs, BamHIlinkers were attached and the fragment was cloned into the unique BamHIsite of pJDB207. From the plasmid with the promoter in acounterclockwise direction, a 1050 base pair SphI fragment was thendeleted (from the SphI site in pJDB207 to the SphI site in the promoterfragment) leaving only a single BamHI site. This plasmid was designatedpALX1.

[0112] Then the PstI site in the B-lactamase gene of pALX1 waseliminated without inactivating. the gene. pALX1 was digested tocompletion with PstI and nuclease S1 to destroy the PstI site, and thensubjected to a partial digestion with PvuI BglI. At the same time a 250base pair PVUI BglI fragment was isolated from pUC8, Vierira, J. andMessing, J. 19 Gene 259 (1982), that contains the corresponding part ofa B-lactamase without a PstI site. This was ligated to the partiallydigested pALX1. In all the ampicillin resistant clones isolated theB-lactamase gene had been restored by incorporating the PUC8 fragment.This plasmid was called pALX2.

[0113] The following oligonucleotide was purchased from Prof. K. Kleppe,University of Bergen, and inserted into the BamHI site of pALX2:     BglII   *      * *   HindIII GATCAGATCTGCAGGATGGATCCAAAGCTT   :initiation codon TCTAGACGTCCTACCTAGGTTTCGAACTAG * : optimal ATG con-      PstI      BamHI text

[0114] Plasmids with the proper orientation were isolated and designatedpALX3.

[0115] Finally, the pALX3 was digested with HindIII and religated todelete a HindIII fragment of 480 base pairs. The resulting vector iscalled pALX4.

[0116] pL4 is a derivative of pALX4 in which the ADHI promoter, isdeleted. pL4 was used as a basis for the insertion of other promoters.pALX4 was first digested with BglII and SalI. The resulting sticky endswere filled-in with the Klenow fragment of DNA polymerase I and 4 dNTPsfollowed by religation. By this treatment the ADHI promoter iseliminated and the BglII site regenerated to give the vector pL4.

EXAMPLE 6 Construction of pαLX5

[0117] The gene for the yeast mating pheromone MFα1 was first cloned byKurjan, J. and Herskowitz, I., “Structure of a Yeast Pheromone Gene(MFα): A Putative-factor Precursor Contains Four Tandem Copies ofMature-factor”. 30 Cell, 93-3-943 (1982). The published sequence wasused to reclone the MFα1 gene. Total yeast DNA from the strain Y288C wasdigested with EcoRI and digestion products in the size range from 1.6 to1.8 kb were isolated from a preparative agarose gel. These were thenligated to dephophorylated EcoRI cleaved pBR322 and used to transform E.coli BJ5183. The resulting clones were screened for MFα1 gene inserts byhybridization to a labeled oligonucleotide of the following composition:

[0118] TGGCATTGGCTGCAACTAAAGC

[0119] DNA from purified positive clones was then used to transform E.coli JA221 from which plasmid DNA was prepared. The plasmid used in thefollowing constructs was pMFα1-1.

[0120] pMFα1-1 was digested with EcoRI, filled-in with the Klenowfragment of DNA polymerase I and 4 dNTPs, phenol extracted and digestedwith BglII. The 1.7 kb MF 1 gene fragment was isolated from an agarosegel. Before inserting it into the yeast shuttle vector, the HindIII siteof pL4 was eliminated by HindIII digestion, Klenow fill-in reaction andreligation to give the pL5 shuttle vector. pL5 was digested with BamHI,filled-in with the Klenow fragment of DNA polymerase I and 4 dNTPs,phenol extracted and digested with BglII. After purification on gel itwas ligated to the MFα1 fragment to give the expression vector pαLX5.

EXAMPLE 7 Construction of pSS LX5-HPTH1

[0121] A 288 base pair BglII XbaI fragment from the pSSHPTH-10 plasmidwas isolated and subcloned in pUC19 using the BamHI and XbaI site ofthis vector. This subclone designated pUC-HPTH, was digested with DpnIand the largest fragment isolated. This fragment was then digested withSalI and the smallest of the two resulting fragments was again isolated,yielding a sticky end on the SalI cut side and a blunt end at the DpnIcut side.

[0122] pαLX5 was digested with HindIII, filled-in with the Klenowfragment of DNA polymerase I and 4 dNTPs, phenol extracted and digestedwith SalI. After purification from gel, it was ligated to the hPTHfragment described above. The resulting clones had the HindIII siteregenerated verifying that the reading frame was correct. This plasmidcalled pSSαLX5-hPTH1. The sequence of the MFα1-hPTH fusion gene is shownin FIG. 6.

EXAMPLE 8 Expression And Secretion Of HPTH In Yeast

[0123] The yeast strain FL200 (, ura3, leu2) was transformed with theplasmids pαLX5 and pSSαLX5-hPTH1 using the spheroplast method. Onetransformant of each kind was grown up in leu⁻ medium and aliquots ofthe cell-free medium were analyzed by SDS-PAGE developed bysilver-staining. Two major bands were seen in the medium from thepSSαLX5-H1 transformant that were not present in the medium from the pLX5 transformant: one band of approximately 9000 daltons, the expectedsize of HPTH, and one band of approximately 16000 daltons that couldcorrespond to an unprocessed MFα1-hPTH fusion product. Both polypeptidesreacted with antibodies against human PTH in a manner identical to thenative hormone.

[0124] The examples are included by way of illustration, but theinvention is not limited thereto. While the above examples are directedto providing a S. cerevisiae which produces and excretes humanparathyroid hormone, the method of the present invention may be appliedto produce a plasmid containing DNA coding for parathyroid hormone fromany species. Further, said plasmid may be inserted into any species ofyeast. The invention thus is not limited to S. cerevisiae.

[0125] The cloned human parathyroid hormone produced by the yeast of thepresent invention has a variety of known and potential uses. Forexample, it is current medical theory that human parathyroid hormonewill be highly effective in treating osteoporosis. Geneticallyengineered parathyroid hormone may be useful in an analytical kit formeasuring parathyroid hormone levels in humans and animals. Humanparathyroid hormone or fragments thereof may also be used for treatmentof humans or animals displaying reduced or pathologically altered bloodcalcium levels. It is anticipated that many other uses will bediscovered when genetically engineered parathyroid hormone is availablein large quantities, for example as a result of the present invention.

EXAMPLE 9 Deletion of the STE 13 Recognition Sequence PositionedN-terminal for the Parathyroid Hormone

[0126] In order to delete the STE13 recognition sequence(Glu-Ala-Glu-Ala) located immediately N-terminal to PTH by site directedin vitro mutagenesis of the fusion gene, a 1495 bp XbaI fragment wasisolated from pSSαLX5-PTH. This contained the α-factor promoter(MFαprom), the α-factor leader sequence (PP) and the human PTH gene(hPTH) including the stop codon. The fragment was subcloned into M13mp19 to give the plasmid p PTHx-M13. An oligonucleotide with thesequence GGATAAAAGATCTGTGAG was made where the first ten nucleotides arecomplementary to the sequence of the α-factor leader in pαPTHx-M13 justproceeding the Glu-Ala-Glu-Ala coding region, and the last eightnucleotides are complementary to the beginning of the human PTHsequence. When this oligonucleotide was annealed to single-stranded DNAprepared from the recombinant phage, the following heteroduplex wasgenerated: oligonucleotide: 5′-GGATAAAAGATCTGTGAG-3′ pPTHx-M13       3′-CCTATTTTCTAGACACTC-5′                             C   A                            T     G [to be removed]                            CCGACTTC translation product . . .AspLysArgSerVal . . . (upper)                  . . .AspLysArgGluAlaGluAlaSerVal . . . (lower)

[0127] After second strand synthesis and ligation with the Klenowfragment of DNA polymerase I and T4 DNA ligase, closed circularheteroduplex DNA was isolated by sedimentation through an alkalinesucrose gradient as described in Carter, P., Bedouelle, H., Waye, M. M.Y., and Winter, G. (1985) “oligonucleotide site-directed mutagenesis inM13. An experimental manual,” MRC Laboratory of Molecular Biology,Cambridge CB2 2QH., the disclosure of which is hereby incorporated byreference. The heteroduplex DNA was used to transform a BMH 71-18 mutLstrain of E. coli defective in mismatch repair (kindly provided by Dr.G. Winter). Positive clones with the looped out sequence3′-CTCCGACTTCGA-5′ deleted were identified by, colony hybridizationusing the mutagenizing oligonucleotide as the probe and by DNAsequencing. The plasmid in these clones was designated pαPTHx-M13ΔEA.

[0128] The α-factor transcription terminator was then inserted into oneof the positive M13 clones as a SalI HindIII fragment isolated frompMFα1, to give a plasmid called pαPTH-M13-ΔEA. The entire expressioncassette between a BamHI and a filled-in EcoRI site was finally isolatedfrom pαPTH-M13-ΔEA and inserted between the BamHI and PvuII site of theyeast shuttle vector YEp24 by the method described in Botstein, D.,Falco, S. C., Stewart, S. E., Brennan, M., Scherer, S., Stinchcomb, D.T., Struhl, K., and Davis, R. W. (1979) Gene 8, 17-24, which is herebyincorporated by reference. This expression plasmid was designatedpSSαUXPTH-ΔEA.

EXAMPLE 10 Conversion of Intact hPTH by Substitution of Lysine withGlutamine at Position 26, Designated PTH_(Q26′)

[0129] In order to change the amino acid at position 26 in the human PTHfrom lysine to glutamine, the fusion gene in pαPTH-M13-ΔEA was furthermodified by in vitro mutagenesis using the “Muta-gene™ in vitromutagenesis kit” obtained from Bio-Rad based on the method of Kunkel;Kunkel, T. A., Roberts, J. D., and Sakour, R. A. (1987) “Rapid andefficient site-specific mutagenesis without phenotypic selection” inMethods of Enzymologi, (Wu, R., and Grossman, L., eds.) vol. 154, pp367-381, which is hereby incorporated by reference. The E. coli strainor CJ236 (dut, ung, thi, rel A; pCJ105 (Cm^(r))) was transformed withthe pαPTH-M13-ΔEA plasmid. The single-stranded DNA that was preparedfrom the phage contained a number of uracils in thymine positions as aresult of the dut mutation (inactivates dUTPase) and the ung mutation(inactivates the repair enzyme uracil N-glycosylase). An oligonucleotidewith the sequence GGCTGCGTCAGAAGCTGC was made where all nucleotidesexcept the ninth are complementary to an internal PTH sequence inpαPTHx-M13. When this oligonucleotide was annealed to thesingle-stranded DNA, the following heteroduplex was generated:                                         C                                        / \oligonucleotide:              5′-GGCTGCGT CAGAAGCTGC-3′paPTH-M13-AEA           3′ . . . CCGACGCA TCTTCGACG . . . 5′                                        \ /                                         T Translation product        .. . LeuArgGlnLysLeu . . . (upper)                            . . .LeuArgLysLysLeu . . . (lower)

[0130] After second strand synthesis and ligation with T4 DNA polymeraseand T4 DNA ligase, the heteroduplex DNA was transformed into the E. colistrain MV1190 ((lac-pro AB), thi, sup E, Δ(sr1-recA)306::Tn10(tet^(r))[F′: tra D36, pro AB, lac I^(q) Z M15]) whichcontains a proficient uracil N-glycosylase. During the repair process inthis host eliminating the uracils in the paternal strand, the in vitrosynthesized strand will serve as a repair template conserving themutation. Positive clones were verified by DNA sequencing. One of thosewere picked and called pαPTH-M13-ΔEA/KQ. The entire expression cassettebetween a BamHI and a filled-in EcoRI site was finally isolated frompαPTH-M13-ΔEA/KQ and inserted between the BamHI and PvuII site of theyeast shuttle vector YEp24. This expression plasmid was designatedpSSαUXPTH-ΔEA/KQ.

EXAMPLE 11 Expression and Secretion of hPTH_(Q26) in Yeast

[0131] The yeast strain BJ1991 (α,Leu2,wa3-52,trpl,pr67-112,pep4-3) wastransformed with the plasmids pSSαUXPTH-ΔEA and pSSαUXPTH-ΔEA/KQ usingthe lithium method. One transformant of each kind was grown in mediumlacking uracil and the cell free medium was analyzed as described below.

EXAMPLE 12 Purification of Heterologous hPTH from Yeast MediumConcentration and Purification by S-Sepharose^(R) Fast Flow

[0132] Samples of cell free yeast medium (1-10 l) (containing 1%Glucose, 2% casamino acid, 134% yeast nitrogen base w/o amino acids, 60mg/ml trp, 180 kg/l) were adjusted to pH 3.0 and run through a 10 ml×10S-Sepharose^(R) (Pharmacia AB) fast flow column, pre-equilibrated with0.1M glycine pH 3.0. The loaded column was eluted by 13 ml 0.1M aceticacid buffered to pH 6.0, followed by 20 ml 0.1M NH₄HC)₃ pH 8.5. Thepeptides eluted from the column were monitored by a Pharmacia opticalunit (Single path monitor UVI, Pharmacia AB, Uppsala, Sweden) at 280 nm,and collected in 2 ml fractions by an LKB 2070 Ultrorac II fractioncollector (LKB, AB, Bromma, Sweden).

EXAMPLE 13 Purification by HPLC

[0133] Collected fractions from S-Sepharose fast flow chromatographywere subjected to further purification by reversed phase HPLC using a 25cm×4.2 cm Vydac protein peptide C18 column (The Separations Group,Hesperia, Calif., USA) and, an LDC gradient mixer, LDC contamertricpumps model I and III with a high pressure mixing chamber and LDCspectromonitor III with variable UV monitor. (LDC Riviera Beach, Fla.,USA). Chromatograms were recorded by a Vitatron 2 channel recorder. Theanalytical conditions were as follows:

[0134] First HPLC purification step:

[0135] Gradient: 35-60%B, 60 min., linear

[0136] A: 0.1% trifluoroacetic acid (TFA)

[0137] B: 70% acetonitril in A (ACN)

[0138] Flow: 1.0 ml/min

[0139] Detection: UV 220 nm

[0140] Second HPLC purification step:

[0141] Same as first step, with the following modification:

[0142] Gradient: 40-45%B 60,min; linear.

EXAMPLE 14 Assessment of the hPTH_(Q26) Product

[0143] This PTH analog was verified to represent the designed product byN-terminal amino acid sequence analysis including amino acid no. 30 andshown to be hPTH identical except for the lysine to glutaminesubstitution at position 26.

[0144] Moreover, the resulting amino acid composition had the expectedalterations, in that the sequence contained one residue less of lysineand one residue more of glutamine.

[0145] Its biological activity was assessed after purification bytesting the effect of synthetically bought human parathyroid hormonefitures in comparison to the recombinant analogue which was equallypotent in stimulating the adenylyl cyclase of bone cell membranes fromrat calveria as well as from an osteosarcoma cell line.

EXAMPLE 15 Additional Examples of Amino Acid Substitutions by SiteSpecific In Vitro Mutagenesis

[0146] By the above method, it is possible to obtain any amino acidsubstitution or sequences of amino acid alterations in the PTH molecule.By use of the “Muta-Gene™ in vitro mutagenesis kit” and syntheticoligonucleotides with the desired sequence corresponding to the aminoacid alteration(s), this may be carried out. Each of theseoligonucleotides can be annealed to the single-stranded DNA in order togenerate a hetroduplex as indicated above.

[0147] Followed by second strand synthesis and ligation with T4 DNApolymerase and T4 DNA ligase, the heteroduplex DNA is transformed intothe E. coli strain MV 1190 with specifications as stated above. In eachof these cases, the repair process in this bacterial host will eliminatethe uracils in the parenteral strands and at the same time, the in vitrosynthesized strand will serve as a repair template whereby theintroduced DNA changes will be conserved. All the positive clones willbe DNA sequenced and the expression cassettes isolated as describedabove and inserted into the yeast shuttle vector YEp 24 fortransformation of Saccharomyces cerevisiae.

[0148] This general approach with the specific alterations as indicated,enables the generation of any desired PTH peptide and PTH like peptide.For example, amino acid substitutions, deletions, insertions orextensions confined within the first 26 amino acids in the N-terminalregion can produce agonists with increased affinity for the PTHreceptors as well as antagonists which bind to the receptor, but arebiologically inactive. The mid-region or the C-terminal part of themolecule is of importance for modifying the binding of PTH to thedifferent receptors in bone cells and the kidney. Changes in either ofthese regions produce an increased or diminished binding affinity to thereceptors in bone cells and the kidney, and this may proposespecialization in binding characteristics so that the PTH derivativecould bind and function only in bone cells or in the kidney, oralteration, i.e., stimulation or blockade, of the biological activity atone or both receptor sites.

[0149] The inventions have been described herein with reference tocertain preferred, embodiments. However, as obvious variations thereonwill become apparent to those skilled in the art, the inventions are notto be considered limited thereto.

EXAMPLE 16 Comparison of the Biological Activity of Human ParathyroidHormone (hPTH 1-84, Bachem Fine Chemicals, Cal. USA) with QPTH

[0150] The purpose of this study was to compare the biological activityof the recombinant QPTH with the standard PTH preparation of Bachemhuman PTH (1-84). We examined the ability of the two agents to inducehypercalcemia in rats. Both the maximum plasma calcium levels as well asthe duration of action was monitored.

[0151] Methods:

[0152] Male Wistar rats (150-200) were parathyroid-ectomized usingelectrocautery 18 hours before the start of the experiment. The animalswere fasted overnight, and anesthetized the next day using hypnormdormicum (0.2 ml per rat). The carotid artery was cannulated usingpolyethylene-50 tubing. The cannula was connected to a syringecontaining Ringers Acetate, 4% bovine serum albumin (BSA), and 25 unitsheparin/ml. Five minutes after injection of 200 Ìl of the heparinizedRingers, a baseline blood sample was drawn (300 Ìl). The animals weretrachesostomized to prevent respiratory failure due to damage to therecurrent laryngeal nerve running through the thyroid gland. The PTH wasthen injected subcutaneously, in a volume of 200 Ìl. Both hPTH and QPTHhad been dissolved into 50 Ìl of 0.01 N acetic acid, allowing at leastone half hour for complete dissolution. After dissolving in the aceticacid, the agents were brought up in 450 Ìl of Ringers Acetate containing1% BSA. Blood samples were then drawn at 1, 2, 3, and 4 hours after theinjection of the PTH. The rats were reheparinized 5 minutes beforedrawing each blood sample using 200 Ìl of the heparinized Ringerssolution.

[0153] The blood samples were centrifuged in a clinical centrifuge for10 minutes, then the plasma was analyzed for calcium using a Cobasautoanalyzer.

[0154] Both the Bachem hPTH and the QPTH induced hypercalcemia in therats to about the same degree and lasting about 2 hours. No significantdifference in the calcium response was seen until 4 hours after theinjections. Then the QPTH maintained the serum calcium better (p<0.05)than synthetic Bachem PTH.

[0155] The zero time plasma calcium (baseline) indicates the time of PTHinjection and was set equal to zero. The changes in plasma calcium fromzero are given as positive or negative values depending on the change(increase or reduction) in the measured values.

Time after injection (hrs) [Calcium mg/100 ml from Baseline]

[0156] Time after injection (hrs) [calcium mg/100 ml from baseline]Median values Preparation 1 2 3 4 hours Bachem hPTH baseline: 6.84 ±0.30 (mg/100 ml) +0.45 +0.30 −0.20 −0.70* QPTH baseline: 7.011 ± 0.29(mg/100 ml) (n = 7) +0.55 +0.25 0.0 −0.50

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[0158] 2/ Norman, A. W., Roth, J., and Orci, L. 1982. The vitamin Dendocrine system—steroid metabolism, hormone receptors, and biologicalresponse (calcium binding proteins). Endocr. Rev. 3, 331-366.

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[0173] 16/ Gautvik, K. M., Teig, V., Halvorsen, J. F., Arnesen, E.,Myhre, L., Heimann, P., and Tollman, R. 1979. Development of sequencespecific radioimmunoassay of human parathyroid hormone and its use inthe diagnosis of hyperparathyroidism. Scand. J. clin. Lab. Invest. 39,469-478

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1 29 1 348 DNA Homo sapiens modified_base (9)..(9) a, t, c or g 1atgathccng cnaargayat ggcnaargtn atgathgtna tgytngcnat htgyttyytn 60acnaarwsng ayggnaarws ngtnaaraar mgnwsngtnw sngarathca rytnatgcay 120aayytnggna arcayytnaa ywsnatggar mgngtngart ggytnmgnaa raarytncar 180gaygtncaya ayttygtngc nytnggngcn ccnytngcnc cnmgngaygc nggnwsncar 240mgnccnmgna araargarga yaaygtnytn gtngarwsnc aygaraarws nytnggngar 300gcngayaarg cngaygtnaa ygtnytnacn aargcnaarw sncartrr 348 2 351 DNA Homosapiens 2 tatgatgata cctgcaaaag acatggctaa agttatgatt gtcatgttggcaatttgttt 60 tcttacaaaa tcggatggga aatctgttaa gaagagatcg tggagtgaaatacagcttat 120 gcataacctg ggaaaacatc tgaactcgat ggagagagta gaatggctgcgtaagaagct 180 gcaggatgtg cacaattttg ttgcccttgg agctcctcta gctcccagagatgctggttc 240 ccagaggccc cgaaaaagga agacaatgtc ttggttgaga gccatgaaaaaagtcttgga 300 gaggcagaca aagctgatgt gaatgtatta actaaagcta aatcccagtg a351 3 432 DNA Homo sapiens modified_base (13)..(13) a, t, c or g 3tatgatgath ccngcnaarg ayatggcnaa rgtnatgath gtnatgytng cnathtgytt 60yytnacnaar wsngayggna arwsngtnaa raarmgnwsn gtnwsngara thcarytnat 120gcayaayytn ggnaarcayy tnaaywsnat ggarmgngtn gartggytnm gnaaraaryt 180ncargaygtn cayaayttyg tngcnytngg ngcnccnytn gcnccnmgng aygcnggnws 240ncarmgnccn mgnaaraarg argayaaygt nytngtngar wsncaygara arwsnytngg 300ngargcngay aargcngayg tnaaygtnyt nacnaargcn aarwsncart rraaatgaaa 360acagatattg tcagagttct gctctagaca gtgtagggca acaatacatg ctgctaattc 420aaagctctat ta 432 4 432 DNA Homo sapiens 4 tatgatgata cctgcaaaagacatggctaa agttatgatt gtcatgttgg caatttgttt 60 tcttacaaaa tcggatgggaaatctgttaa gaagagatct gtgagtgaaa tacagcttat 120 gcataacctg ggaaaacatctgaactcgat ggagagagta gaatggctgc gtaagaagct 180 gcaggatgtg cacaattttgttgcccttgg agctcctcta gctcccagag atgctggttc 240 ccagaggccc cgaaaaaaggaagacaatgt cttggttgag agccatgaaa aaagtcttgg 300 agaggcagac aaagctgatgtgaatgtatt aactaaagct aaatcccagt gaaaatgaaa 360 acagatattg tcagagttctgctctagaca gtgtagggca acaatacatg ctgctaattc 420 aaagctctat ta 432 5 432DNA Homo sapiens CDS (5)..(349) 5 tatg atg ata cct gca aaa gac atg gctaaa gtt atg att gtc atg ttg 49 Met Ile Pro Ala Lys Asp Met Ala Lys ValMet Ile Val Met Leu 1 5 10 15 gca att tgt ttt ctt aca aaa tcg gat gggaaa tct gtt aag aag aga 97 Ala Ile Cys Phe Leu Thr Lys Ser Asp Gly LysSer Val Lys Lys Arg 20 25 30 tct gtg agt gaa ata cag ctt atg cat aac ctggga aaa cat ctg aac 145 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu GlyLys His Leu Asn 35 40 45 tcg atg gag aga gta gaa tgg ctg cgt aag aag ctgcag gat gtg cac 193 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu GlnAsp Val His 50 55 60 aat ttt gtt gcc ctt gga gct cct cta gct ccc aga gatgct ggt tcc 241 Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp AlaGly Ser 65 70 75 cag agg ccc cga aaa aag gaa gac aat gtc ttg gtt gag agccat gaa 289 Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser HisGlu 80 85 90 95 aaa agt ctt gga gag gca gac aaa gct gat gtg aat gta ttaact aaa 337 Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu ThrLys 100 105 110 gct aaa tcc cag tgaaaatgaa aacagatatt gtcagagttctgctctagac 389 Ala Lys Ser Gln 115 agtgtagggc aacaatacat gctgctaattcaaagctcta tta 432 6 115 PRT Homo sapiens 6 Met Ile Pro Ala Lys Asp MetAla Lys Val Met Ile Val Met Leu Ala 1 5 10 15 Ile Cys Phe Leu Thr LysSer Asp Gly Lys Ser Val Lys Lys Arg Ser 20 25 30 Val Ser Glu Ile Gln LeuMet His Asn Leu Gly Lys His Leu Asn Ser 35 40 45 Met Glu Arg Val Glu TrpLeu Arg Lys Lys Leu Gln Asp Val His Asn 50 55 60 Phe Val Ala Leu Gly AlaPro Leu Ala Pro Arg Asp Ala Gly Ser Gln 65 70 75 80 Arg Pro Arg Lys LysGlu Asp Asn Val Leu Val Glu Ser His Glu Lys 85 90 95 Ser Leu Gly Glu AlaAsp Lys Ala Asp Val Asn Val Leu Thr Lys Ala 100 105 110 Lys Ser Gln 1157 874 DNA Artificial Sequence Description of Artificial SequenceMF-alpha1- hPTH fusion gene 7 agtgcaagaa aaccaaaaag caacaacaggttttggataa gtacatatat aagagggcct 60 tttgttccca tcaaaaatgt tactgttcttacgattcatt tacgattcaa gaatagttca 120 aacaagaaga ttacaaacta tcaatttcatacacaatata aacgaccaaa agaatgagat 180 ttccttcaat ttttactgca gttttattcgcagcatcctc cgcattagct gctccagtca 240 acactacaac agaagatgaa acggcacaaattccggctga agctgtcatc ggttactcag 300 atttagaagg ggatttcgat gttgctgttttgccattttc caacagcaca aataacgggt 360 tattgtttat aaatactact attgccagcattgctgctaa agaagaaggg gtatctttgg 420 ataaaagaga ggctgaagct wsngtnwsngarathcaryt natgcayaay ytnggnaarc 480 ayytnaayws natggarmgn gtngartggytnmgnaaraa rytncargay gtncayaayt 540 tygtngcnyt nggngcnccn ytngcnccnmgngaygcngg nwsncarmgn ccnmgnaara 600 argargayaa ygtnytngtn garwsncaygaraarwsnyt nggngargcn gayaargcng 660 aygtnaaygt nytnacnaar gcnaarwsncartrraaatg aaaacagata ttgtcagagt 720 tctgctctag agtcgacttt gttcccactgtacttttagc tcgtacaaaa tacaatatac 780 ttttcatttc tccgtaaaca acctgttttcccatgtaata tccttttcta tttttcgttt 840 cgttaccaac tttacacata ctttatatagctat 874 8 874 DNA Artificial Sequence Description of ArtificialSequence MF-alpha1- hPTH fusion gene 8 agtgcaagaa aaccaaaaag caacaacaggttttggataa gtacatatat aagagggcct 60 tttgttccca tcaaaaatgt tactgttcttacgattcatt tacgattcaa gaatagttca 120 aacaagaaga ttacaaacta tcaatttcatacacaatata aacgaccaaa agaatgagat 180 ttccttcaat ttttactgca gttttattcgcagcatcctc cgcattagct gctccagtca 240 acactacaac agaagatgaa acggcacaaattccggctga agctgtcatc ggttactcag 300 atttagaagg ggatttcgat gttgctgttttgccattttc caacagcaca aataacgggt 360 tattgtttat aaatactact attgccagcattgctgctaa agaagaaggg gtatctttgg 420 ataaaagaga ggctgaagct tctgtgagtgaaatacagct tatgcataac ctgggaaaac 480 atctgaactc gatggagaga gtagaatggctgcgtaagaa gctgcaggat gtgcacaatt 540 ttgttgccct tggagctcct ctagctcccagagatgctgg ttcccagagg ccccgaaaaa 600 aggaagacaa tgtcttggtt gagagccatgaaaaaagtct tggagaggca gacaaagctg 660 atgtgaatgt attaactaaa gctaaatcccagtgaaaatg aaaacagata ttgtcagagt 720 tctgctctag agtcgacttt gttcccactgtacttttagc tcgtacaaaa tacaatatac 780 ttttcatttc tccgtaaaca acctgttttcccatgtaata tccttttcta tttttcgttt 840 cgttaccaac tttacacata ctttatatagctat 874 9 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 9 ggataaaaga tctgtgag 18 10 18 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 10ggctgcgtca gaagctgc 18 11 17 PRT Influenza virus 11 Met Lys Ala Lys LeuLeu Val Leu Leu Thr Ala Phe Val Ala Thr Asp 1 5 10 15 Ala 12 18 PRT Homosapiens 12 Met Arg Ser Leu Leu Ile Leu Val Leu Cys Phe Leu Pro Leu AlaAla 1 5 10 15 Leu Gly 13 19 PRT Saccharomyces cerevisiae 13 Met Arg PhePro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala LeuAla 14 18 PRT Kluyveromyces lactis 14 Met Asn Ile Phe Tyr Ile Phe LeuPhe Leu Ser Phe Val Gln Gly Thr 1 5 10 15 Arg Gly 15 24 DNA ArtificialSequence Description of Artificial Sequence Probe 15 tactatggacgttttctgta ccga 24 16 24 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 16 ctcaagacga gatctgtcacatcc 24 17 30 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 17 gatcagatct gcaggatgga tccaaagctt 30 18 30DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 18 gatcaagctt tggatccatc ctgcagatct 30 19 22 DNAArtificial Sequence Description of Artificial Sequence Probe 19tggcattggc tgcaactaaa gc 22 20 4 PRT Homo sapiens 20 Glu Ala Glu Ala 121 30 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 21 ctcacagaag cttcagcctc tcttttatcc 30 22 5PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 22 Asp Lys Arg Ser Val 1 5 23 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 23 Asp Lys Arg GluAla Glu Ala Ser Val 1 5 24 12 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 24 agcttcagcc tc 12 25 18DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 25 ggctgcgtca gaagctgc 18 26 19 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 26ggctgcgtcc agaagctgc 19 27 18 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 27 gcagcttctt acgcagcc 1828 5 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 28 Leu Arg Gln Lys Leu 1 5 29 5 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 29 Leu ArgLys Lys Leu 1 5

We claim:
 1. Essentially pure recombinant hPTH.
 2. Substantially puresynthetic hPTH.
 3. Substantially pure recombinant hPTH.
 4. Thesubstantially pure recombinant hPTH of claim 3 wherein said hPTH is atleast about 90% pure.
 5. The substantially pure recombinant hPTH ofclaim 4 wherein said hPTH is at least about 95% pure.
 6. Substantiallypure recombinant hPTH which is resistant to degradation by a KEX2 likeproteolytic enzyme.
 7. A substantially pure hPTH derivative which isboth intact and exhibits native biological activity.
 8. A substantiallypure hPTH derivative which is resistant to degradation by a KEX2 likeproteolytic enzyme and which is both intact and exhibits nativebiological activity.
 9. A genetically engineered microorganism capableof expressing an intact hPTH.
 10. The microorganism of claim 9 whereinsaid organism is yeast.
 11. A substantially pure intact hPTH, obtainedby expression and secretion of said hPTH from a geneticallyengineered-microorganism.
 12. The substantially pure intact hPTH ofclaim 11, wherein said hPTH is resistant to degradation by a KEX2 likeproteolytic enzyme.
 13. The substantially pure intact hPTH of claim 11,which is obtained by a purification step after expression and secretion.14. The substantially pure intact hPTH of claim 12, which is obtained bya purification step after expression and secretion.
 15. Thesubstantially pure intact hPTH of claim 11, wherein said geneticallyengineered microorganism is yeast.
 16. A substantially pure intact hPTHderivative, obtained by expression and secretion of said hPTH from agenetically engineered microorganism.
 17. The substantially pure intacthPTH derivative of claim 16, wherein said hPTH is resistant todegradation by a KEX2 like proteolytic enzyme.
 18. The substantiallypure intact hPTH derivative of claim 16, which is obtained by apurification step after expression and secretion.
 19. The substantiallypure intact hPTH derivative of claim 17, which is obtained by apurification step after expression and secretion.
 20. The substantiallypure intact hPTH of claim 16, wherein said genetically engineeredmicroorganism is yeast.